Hydroxylation of aromatic rings is an important reaction used for the preparation of many valuable compounds including L-DOPA for the treatment of Parkinson's disease, benzylisoquinoline alkaloids, and melatonin. Compared with chemical reaction which frequently uses metallic oxidants in organic solvent, hydroxylation of aromatic ring by microorganisms is an interesting and promising method to synthesize the desired products in a single-step with a high regioselectivity and under mild conditions. Microbial aromatic hydroxylation is involved in the aerobic metabolism of aromatic compounds and mostly performed by oxygenases and tyrosinases during the degradation process either to relieve the toxicity or to metabolize them into organic acid to use as carbon sources.
Tyrosinase is an oxidoreductase belongs to type-3 copper protein which includes hemocyanins as an oxygen carrier. (Olivares, 2009; Robb, 1984) This enzyme involves multiple oxidation reaction of L-tyrosine using molecular oxygen as oxidant; the first oxidation step is o-hydroxylation of L-tyrosine to L-DOPA and is known to be the slowest step, and the second oxidation step is the production of o-quinone from o-diphenol which is fast and followed by non-enzymatic reaction to dopachrome, a colored intermediate to melanin pathway. Microbial conversion of tyrosine to L-DOPA is slow process, and the over-oxidation to ortho-quinone is hard to avoid when tyrosinase is used. The use of reducing agent such as ascorbic acid adds more step for the purification of the product from fermentation broth.
L-DOPA is an important compound to living cells, especially in animal since it is used as a precursor for many neurotransmitters, and in animal brain, L-DOPA was synthesized by tyrosine hydroxylase (TH) with tetrahydrobiopterin (BH4) as a cofactor. (Kappock, Chem. Rev. 1996; Fitzpatrick, Ann Rev Biochem 1999; Daubner, Arch Biochem Biophys 2011) The use of pterin cofactor during the oxidation step is unique feature of TH and related enzyme such as phenylalanine hydroxylase (PAH) and tryptophan hydroxylase (TPH), (Pribat, J. Bacteriol. 2010) and this helps to prevent over-oxidation of L-tyrosine to o-quinone product which is a problem in microbial L-DOPA production by tyrosinase (Maass, 2003). However, the application of TH enzyme to microbial metabolic engineering has not been reported due to the unavailability of the coenzyme BH4 in microbes. BH4 is a unique co-factor found in animal and no bacterial system has been reported to use BH4 for biosynthesis of L-DOPA or related metabolites.